Composition of polymers

ABSTRACT

Compositions are described comprising a of a block copolymer having an overall ionic charge and in which one of the blocks has pendant zwitterionic groups and a biologically active compound having a charge opposite that of the polymer. The polymer is preferably a linear diablock copolymer, preferably having a low polydispersity, such as a (tertiary amine group containing monomer) block-(zwitterionic monomer) copolymer. Suitable cationic monomers are dialkyl aminoalkyl(alk)acrylates and -acrylamides and suitable zwitterionic monomers are phosphorylcholine group containing acrylate monomers such as 2-methacyloyloxyetyl-2 1 -trimethyl ammonium ethyl phosphate liner salt. The biologically active compound is generally polyionic and is for instance a nucleic acid, such as DNA, especially plasmid DNA.

The present invention relates to compositions comprising polymers andbiologically active compounds, especially polymeric drug deliverysystems.

DNA delivery has been a flagship in non-viral gene delivery because thepromise of therapeutic DNA delivery as a potential cure for many geneticdiseases has stimulated much interest over the past decade. Withunacceptable immune responses and other adverse events recently reportedfor viral delivery, nonviral gene delivery becomes even more attractive.However, the main limitation with non-viral delivery is the inefficienttransfection, caused mainly by the poor transport of DNA across cellmembranes. Various cationic polyelectrolytes have shown promisingeffects in facilitating gene delivery as these polymers readilyconjugate with DNA to neutralize the net negative charges from DNAmolecules. However, recent research has indicated that successfulpolymeric candidates must satisfy a set of requirements. First, althoughneutralization helps to mediate negative charges in DNA and improve thetransport, the polymer must not destabilize the helical structures sothat its bioactivity is lost. In addition, it must not imposecytotoxicity to cells either. Cationic polymers such as poly-L-lysine(PLL), polyethylenimine (PEI) and polyamidoamine dendrimer all readilyfrom molecular conjugates with DNA. Unfortunately, high toxicity hasbeen reported from these polymers, which is associated with thedissociation of protons on the primary and secondary amine side chains(the so called proton sponge effect). This event occurs in the endosomalcompartment of cell, which activates the complement system leading tocell death. Second, Langer et al have demonstrated that for effectivegene delivery, the size of conjugated particles must not be over 150 nmfor them to be engulfed by cells Pack, Putnam, Langer, Biotech. Bioeng.2000, 67, 217. Third, the conjugated particles must be readilydispersable in aqueous solution. Unstable aggregates are difficult toadminister and are rapidly cleared from systemic administration. Theseconditions together with the level of the high cost in the synthesis ofsome of the cationic polymers mean that few existing polymers can meetthis set of requirements.

Copolymers of tertiary amine alkyl methacrylates with polyethyleneglycol have been investigated for their potential to serve as vectorsfor gene therapy, by Rungfardthong, U. et al J. Contr. Rel. (2001),73(2-3), 359-380. Polymers investigated included low polydispersityblock copolymers as well as comb polymers formed by statisticalcopolymerisation of the tertiary amine alkyl methacrylate and amono-methacrylated oligo(ethyleneglycol) monomer. The incorporation ofthe PEG moieties enabled colloidally stable complexes of polymer and DNAto be generated. In vitro transfection experiments showed sometransfection took place, albeit that lower levels than a controlpoly-L-lysine system.

In WO-A-99/06055 block copolymers comprising a non-ionic block and acationic block are used to deliver nucleic acids. The non-ionic blockmay comprise polyacrylamide. The cationic block may comprisepolyethylene imine, or a polyimine polymer formed from dibromobutane andN-(3-aminopropyl)-1,3-propane diamine, or a lysine polymer or copolymer(with alanine). Polynucleotide complexed with the copolymers wereprotected from nuclease attack, and were successful in in vivo and invitro transfection experiments.

Cationic drugs have been delivered using systems based on anionicpolymers, for instance block copolymers comprising non-ionic blocks andanionic blocks. Govender, T. et al in J. Contr. Rel. (2001), 75(3),249-258 describe non-covalent interactions between a poly(aspartic acid)poly(ethylene glycol) block copolymer with diminazene aceturate, a lowmolecular weight cationic drug.

Bronich, T. K. et al in Langmuir (2000), 16(2), 481-489, describe blockcopolymers of polyethylene oxide and poly(sodium methacrylate) withcationic surfactants such as cetylpyridinium bromide. The complexesformed stable dispersions with particle sizes in the range 100 to 200nm. The authors describe the effect of changing the block length of thePEO block, and of the sodium methacrylate block on the properties of thedispersion.

Similar disclosures are in WO-A-98156348 and WO-A-98/56334, In ourearlier patent publications WO-A-98/22516 and WO-A-9822517 we describepolymers, primarily used for coating substrates, having pendant cationicand zwitterionic groups, used in conjunction with anionic biologicallyactive materials such as anionic mucopolysaccharides, especiallyheparin. Although the polymer is generally coated onto a substrate, andthe coated substrate subsequently contacted with the anionic active, itis also suggested that the polymer and active may be premixed in acompatible solvent to form a coating solution.

In our earlier publications WO-A-00/28920 and WO-A-00/29481, we describepolyion complexes formed of oppositely charged polyelectrolytes, atleast one of which has pendant zwitterionic groups. The polyioncomplexes may be used as drug delivery depots, although there are noexamples of selection of specific polymers for use with specificactives.

In Langmuir (2000) 16, 5980-5986 Styrkas D. A. et al describe adsorptionat a solid-liquid interface of low polydispersity block copolymersformed of a tertiary amino alkylmethacrylate block and asulphobetaine-group containing block. The adsorption showed pH-dependenteffects which the authors compared to the pH dependent effects ofmicelle formation of these block copolymers described in earlier work byBütün, V. et al. in J. Mater. Chem. (1997), 7, 1693.

A new composition according to the invention comprises a block copolymerhaving an overall ionic charge and associated with the polymer abiologically active compound having a charge opposite that of thepolymer and is characterised in that the block copolymer comprises atleast one block which has pendant zwitterionic groups and at least oneblock which comprise ionic groups to confer said overall ionic charge.

The invention is of most value where the biologically active compound isanionic, preferably polyanionic, in nature. The invention is of mostvalue where the active compound is a nucleic acid, for instance anoligonucleotide, having 5 to 50 base residues usually of DNA. Forinstance the oligonucleotide may be an active anti-sense molecule. Thenucleic acid may alternatively be a single strand RNA molecule or asingle or double strand DNA molecule. Double stranded DNA may, forinstance, comprise genes encoding useful products, especially a plasmid,including control sequences enabling it to be transcribed and translatedwhen transfected into a cell. The invention is thus usefully a genedelivery system. Other anionic actives may be saccharide-containingcompounds, proteins or peptides and amphiphilic anionic compounds suchas retinoic acid and derivatives.

The invention may also be useful where the biologically active compoundis a cationic drug, especially a polycationic drug or an amphiphiliccationic drug. Examples are cetyl and other long chain alkyl-pyridiniumcompounds, anaesthetics, such as procaine-HCl, rhodamine probes, and lowmolecular weight drugs such as mexilitine, amiloride HCl, diminazeneaceturate and amikicin sulphate.

The composition of the invention is preferably in the form of an aqueouscomposition or a non-aqueous composition which may be made up to form anaqueous composition by addition of water. In the invention the term“associated with” in relation to the interaction between the polymer andthe biologically active compound means that the polymer and the activeare electrostatically bound to one another. They are not covalentlybound. More preferably the composition comprises polymer andbiologically active compound associated with one another in the form ofparticles having an average diameter of less than 200 nm, preferablyless than 150 nm Preferably the particles are in suspension where thecomposition is the preferred aqueous liquid. Particles of size less thanthe indicated maximum, are capable of being taken up by cells, so thatthe biologically active compounds may be delivered intracellularly. Suchparticles may also be stabilized against settlement in an aqueouscomposition. Such a composition thus retains useful rheology, enablingit to be handled by usual liquid handling techniques, without having tobe thickened or gelled to stabilise the particles against settlement.

The particle size may depend upon the molecular size of the biologicallyactive compound and/or of the copolymer. It will also depend upon otherfeatures of the copolymer, for instance the nature of the monomers fromwhich the polymer is formed. Preferably the copolymer has a molecularweight (weight average) less than 500,000, preferably less than 100,000,for instance 50,000 Da.

Generally the zwitterionic block is formed from ethylenicallyunsaturated monomers including a zwitterionic monomer having the generalformulaYBX  Iin which

-   -   Y is an ethylenically unsaturated group selected from        H₂C═CR—CO-A-, H₂C═CR—C₆H₄-A¹-, H₂C═CR—CH₂A², R²O—CO—CR═CR—CO—O,        RCH═CH—CO—O—, RCH═C(COOR²)CH₂—CO—O,

A is —O— or NR¹;

A¹ is selected from a bond, (CH₂)_(l)A² and (CH₂)_(l)SO₃— in which l is1 to 12;

A² is selected from a bond, —O—, O—CO—, CO—O, CO—NR¹—, —NR¹—CO,O—CO—NR¹—, NR¹—CO—O—;

R is hydrogen or C₁₋₄ alkyl;

R¹ is hydrogen, C₁₋₄ alkyl or BX;

R² is hydrogen or C₁₋₄ alkyl;

B is a bond, or a straight branched alkenediyl, alkylene oxaalkylene, oralkylene (oligooxalkylene) group, optionally containing one or morefluorine substituents;

X is a zwitterionic group.

Preferably X is an ammonium, phosphonium, or sulphonium phosphate orphosphonate ester zwitterionic group, more preferably a group of thegeneral formula II

in which the moieties A³ and A⁴, which are the same or different, are—O—, —S—, —NH— or a valence bond, preferably —O—, and W⁺ is a groupcomprising an ammonium, phosphonium or sulphonium cationic group and agroup linking the anionic and cationic moieties which is preferably aC₁₋₁₂-alkanediyl group,

preferably in which W⁺ is a group of formula—W¹—N⁺R³ ₃, —W¹—P⁺R⁴ ₃, —W¹—S⁺R⁴ ₂ or —W¹-Het⁺ in which:

W¹ is alkanediyl of 1 or more, preferably 2-6 carbon atoms optionallycontaining one or more ethylenically unsaturated double or triple bonds,disubstituted-aryl (arylene), alkylene arylene, arylene alkylene, oralkylene aryl alkylene, cycloalkanediyl, alkylene cycloalkyl, cycloalkylalkylene or alkylene cycloalkyl alkylene, which group W¹ optionallycontains one or more fluorine substituents and/or one or more functionalgroups; and

either the groups R³ are the same of different and each is hydrogen oralkyl of 1 to 4 carbon atoms, preferably methyl, or aryl, such asphenyl, or two of the groups R³ together with the nitrogen atom to whichthey are attached form an aliphatic heterocyclic ring containing from 5to 7 atoms, or the three groups R³ together with the nitrogen atom towhich they are attached as heteroaromatic ring having 5 to 7 atoms,either of which rings may be fused with another saturated or unsaturatedring to form a fused ring structure containing from 5 to 7 atoms in eachring, and optionally one or more of the groups R³ is substituted by ahydrophilic functional group, and

the groups R⁴ are the same or different and each is R³ or a group OR³,where R³ is as defined above; or

Het is an aromatic nitrogen-, phosphorus or sulphur-, preferablynitrogen-, containing ring, for example pyridine.

Monomers in which X is of the general formula in which W⁺ is W¹N^(⊕)R³ ₃may be made as described in our earlier specification WO-A-9301221.Phosphonium and sulphonium analogues are described in WO-A-9520407 andWO-A-9416749.

Generally a group of the formula II has the preferred general formulaIII

where the groups R⁵ are the same or different and each is hydrogen orC₁₋₄ alkyl, and m is from 1 to 4, in which preferably the groups R⁵ arethe same is preferably methyl.

In phosphobetaine based groups, X may have the general formula IV

in which A⁵ is a valence bond, —O—, —S— or —NH—, preferably —O—;

R⁶ is a valence bond (together with A⁵) or alkanediyl, —C(O)alkylene- or—C(O)NH alkylene preferably alkanediyl, and preferably containing from 1to 6 carbon atoms in the alkanediyl chain;

W² is S, PR⁷ or NR⁷;

the or each group R⁷ is hydrogen or alkyl of 1 to 4 carbon atoms or thetwo groups R⁷ together with the heteroatom to which they are attachedform a heterocyclic ring of 5 to 7 atoms;

R⁸ is alkanediyl of 1 to 20, preferably 1 to 10, more preferably 1 to 6carbon atoms;

A⁶ is a bond, NH, S or O, preferably O; and

R⁹ is a hydroxyl, C₁₋₁₂ alkyl, C₁₋₁₂ alkoxy, C₇₋₁₈ aralkyl,C₇₋₁₈-aralkoxy, C₆₋₁₈ aryl or C₆₋₁₈ aryloxy group.

Monomers comprising a group of the general formula IV may be made bymethods as described in JP-B-03-031718, in which an amino substitutedmonomer is reacted with a phospholane.

In compounds comprising a group of the general formula IV, it ispreferred that

A⁵ is a bond;

R⁶ is a C₂₋₆ alkanediyl;

W² is NR⁷:

each R⁷ is C₁₋₄ alkyl;

R⁸ is C₂₋₆ alkanediyl;

A⁶ is O; and

R⁹ is C₁₋₄ alkoxy.

Alternatively X may be a zwitterion in which the anion comprises asulphate, sulphonate or carboxylate group.

One example of such a group is a sulphobetaine group, of the generalformula V

where the groups R¹⁰ are the same or different and each is hydrogen orC₁₋₄ alkyl and s is from 2 to 4.

Preferably the groups R¹⁰ are the same. It is also preferable that atleast one of the groups R¹⁰ is methyl, and more preferable that thegroups R¹⁰ are both methyl.

Preferably s is 2 or 3, more preferably 3.

Another example of a zwitterionic group having a carboxylate group is anamino acid moiety in which the alpha carbon atom (to which an aminegroup and the carboxylic acid group are attached) is joined through alinker group to the backbone of the biocompatible polymer. Such groupsmay be represented by the general formula VI

in which A⁷ is a valence bond, O—, —S— or —NH—, preferably —O—,

R¹¹ is a valence bond (optionally together with A⁷) or alkanediyl,—C(O)alkylene- or —C(O)NHalkylene, preferably alkanediyl and preferablycontaining from 1 to 6 carbon atoms; and

the groups R¹² are the same or different and each is hydrogen or alkylof 1 to 4 carbon atoms, preferably methyl, or two or three of the groupsR¹², together with the nitrogen to which they are attached, form aheterocyclic ring of from 5 to 7 atoms, or the three group R¹² togetherwith the nitrogen atom to which they are attached form a fused ringheterocyclic structure containing from 5 to 7 atoms in each ring.

Another example of a zwitterion having a carboxylate group is a carboxybetaine —N^(⊖)(R¹³)₂(CH₂)_(r)COO^(⊖) in which the R¹³ groups are thesame or different and each is hydrogen or R₁₋₄ alkyl and r is 2 to 6,preferably 2 or 3.

In the zwitterionic monomer of the general formula I it is preferredthat the ethylenic unsaturated group Y is H₂C═CR—CO-A-. Such acrylicmoieties are preferably methacrylic, that is in which R is methyl, oracrylic, in which R is hydrogen. Whilst the compounds may be(meth)acrylamido compounds (in which A is NR¹), in which case R¹ ispreferably hydrogen, or less preferably, methyl, most preferably thecompounds are esters, that is in which A is O.

In monomers of the general formula I, especially where Y is thepreferred (alk)acrylic group, B is most preferably an alkanediyl group.Whilst some of the hydrogen atoms of such group may be substituted byfluorine atoms, preferably B is an unsubstituted alkanediyl group, mostpreferably a straight chain group having 2 to 6 carbon atoms.

A particularly preferred zwitterionic monomer is2-methacryloyloxyethyl-2′-trimethylammonium ethyl phosphate inner salt(MPC).

The ionic block may be formed of condensation polymers, such aspolyethers, polyesters, polyamides, polyanhydrides polyurethanes,polyimines, polypeptides, polyureas, polyacetals, polysaccharides orpolysiloxanes. One example of a suitable ionic block ispolyethylenimine, copolymers of which with polyalkylene oxides have beeninvestigated as drug delivery components. Preferably, however, the blockis formed by radical polymerisation of ethylenically unsaturatedmonomers.

It is preferred that the ionic block comprise pendant cationic oranionic groups. Cationic pendant groups are, for instance, primary,secondary or tertiary amines, capable of being protonated at pH's in therange 4 to 10. Alternatively a cationic group may be a phosphine. Ananionic group may be a phosphate, phosphonate, sulphate, sulphonate,carbonate or preferably carboxylate group.

Suitable ionic monomers from which the ionic block is formed have thegeneral formula VIIY¹B¹Q  VIIin which Y¹ is selected from H₂C═CR¹⁴—CO-A⁸-, H₂C═CR¹⁴—C₅H₄-A⁹-,H₂C═CR¹⁴—CH₂A¹⁰, R¹⁰O—CO—CR¹⁴═CR¹⁴—CO—O, R¹⁴CH═CH—CO—O—,R¹⁴CH═C(COOR¹⁶)CH₂—CO—O,

A⁸ is —O— or NR¹⁵;

A⁹ is selected from a bond, (CH₂)_(q)A¹⁰ and (CH₂)_(q)SO₃— in which q is1 to 12;

A¹⁰ is selected from a bond, —O—, —O—CO—, —CO—O, —CO—NR¹⁵—, —NR¹⁵CO—,—O—CO—NR¹⁵—, —NR¹⁵—CO—O—;

R¹⁴ is hydrogen or C₁₋₄ alkyl;

R¹⁵ is hydrogen, C₁₋₄ alkyl or B¹Q;

R¹⁶ is hydrogen or C₁₋₄ alkyl;

B¹ is a bond, or a straight branched alkanediyl, alkylene oxaalkylene,or alkylene (oligooxalkylene) group, optionally containing one or morefluorine substituents; and

Q is an ionic or ionisable moiety.

By the term ionic monomer, we include ionisable monomers. In thecomposition where the polymer is associated with the active, the groupderived from the ionic monomer should be ionised. Ionisation may takeplace after polymerisation, however.

Examples of cationic or cationisable groups Q have the formula —NR¹⁷_(p), —PR¹⁷ _(p) and SR¹⁷ _(r), in which p is 2 or 3, r is 1 or 2, thegroups R¹⁷ are the same or different and each is selected from the groupconsisting of hydrogen, C₁₋₂₄ alkyl and aryl, or two of the groups R¹⁷together with the heteroatom to which they are attached from a 5 to 7membered heterocyclic ring or three R¹⁷ groups together with theheteroatom to which they are attached form a 5 to 7 memberedheteroaromatic ring, either of which rings may be fused to another 5 to7 membered saturated or unsaturated ring, and any of the R¹⁷ groups maybe substituted by amino or hydroxyl groups or halogen atoms.

Preferably Q is NR¹⁷ ₂ where R¹⁷ is C₁₋₁₂-alkyl. Preferably both R¹⁷'sare the same. Particularly useful results have been achieved where thegroups R¹⁷ are C₁₋₄ alkyl, especially ethyl.

Where the monomer of the general formula VII provides anionic oranionisable groups, for instance carboxylate, or carboxylic acid group,B¹ is a bond, A⁸ is —O— and Q is hydrogen. Alternative monomersproviding carboxylate or carboxylic acid moieties have B¹ as other thana bond, and Q as a carboxylate or carboxylic acid group. Where Q is ananionic or anionisable group other than carboxylate or carboxylic acidgroup, then B¹ is other than a bond, and Q is a group of general formulaVIII

in which A¹¹ is a bond, NH, S or O, preferably O; and

R¹⁸ is a hydroxyl, C₁₋₁₂alkyl, C₁₋₁₂-alkoxy, C₇₋₁₈-aralkyl,C₇₋₁₈-arylkoxy, C₆₋₁₈-aryl or C₆₋₁₈-aryloxy group.

Alternatively Q may be a group SO₃ ⁻.

Preferably Y¹ is H₂C═CR¹⁴COA⁸ where R¹⁴ is H or CH₃ and A⁸ is O or NH.

B¹ is preferably C₂₋₆-alkanediyl, preferably (CH₂)₂₋₆.

Either or both the zwitterionic and ionic blocks may include comonomers,for instance to provide functionality, control over hydrophobicity,control over pH sensitivity, pK_(A) or pK_(B) as the case may be, or asgeneral diluents. For instance comonomers providing functionality may beuseful to provide conjugation of pendant groups following polymerisationand/or micelle formation, to targeting moieties, or to provide forconjugation between the biologically active molecule and the polymer.Alternatively, functional groups may allow for crosslinking of thepolymer following micelle formation, to confer increased stability onthe micellar structure.

Examples of suitable comonomers are compounds of the general formula IX

in which R¹⁹ is selected from hydrogen, halogen, C₁₋₄ alkyl and groupsCOOR²³ in which R²³ is hydrogen and C₁₋₄ alkyl;

R²⁰ is selected from hydrogen, halogen and C₁₋₄ alkyl;

R²¹ is selected from hydrogen, halogen, C₁₋₄ alkyl and groups COOR²³provided that R¹⁹ and R²¹ are not both COOR²³, and

R²² is a C₁₋₁₀ alkyl, a C₁₋₂₀ alkoxycarbonyl, a mono- or di-(C₁₋₂₀alkyl) amino carbonyl, a C₆₋₂₀ aryl (including alkaryl) a C₇₋₂₀ aralkyl,a C₆₋₂₀ aryloxycarbonyl, a C₁₋₂₀-aralkyloxycarbonyl, a C₆₋₂₀ arylaminocarbonyl, a C₇₋₂₀ aralkyl-amino, a hydroxyl or a C₂₋₁₀ acyloxy group,any of which may have one or more substituents selected from halogenatoms, alkoxy, oligo-alkoxy, aryloxy, acyloxy, acylamino, amine(including mono and dialkyl amino and trialkylammonium in which thealkyl groups may be substituted), carboxyl, sulphonyl, phosphoryl,phosphino, (including mono and di-alkyl phosphine andtri-alkylphosphonium), zwitterionic, hydroxyl groups, vinyloxycarbonyland other vinylic or allylic substituents, and reactive silyl orsilyloxy groups, such as trialkoxysilyl groups;

or R²² and R²¹ or R²² and R²⁰ may together form —CONR²⁴CO in which R²⁴is a C₁₋₂₀ alkyl group.

It is preferred for at least two of the groups R¹⁹, R²⁰, R²¹ and R²² tobe halogen or, more preferably, hydrogen atoms. Preferably R¹⁹ and R²⁰are both hydrogen atoms. It is particularly preferred that compound ofgeneral formula IX be a styrene-based or acrylic based compound. Instyrene based compounds R²² represents an aryl group, especially asubstituted aryl group in which the substituent is an amino alkyl group,a carboxylate or a sulphonate group. Where the comonomer is an acrylictype compound, R²² is an alkoxycarbonyl, an alkyl amino carbonyl, or anaryloxy carbonyl group. Most preferably in such compounds R²² is aC₁₋₂₀-alkoxy carbonyl group, optionally having a hydroxy substituent.Acrylic compounds are generally methacrylic in which case R²¹ is methyl.

Preferably the comonomer is a non-ionic comonomer, such as a C₁₋₂₄alkyl(alk)-acrylate or acrylamide, mono- ordi-hydroxy-C₁₋₆-alkyl(alk)-acrylate, or -acrylamide, oligo(C₂₋₃ alkoxy)C₂₋₁₈-alkyl(alk)-acrylate, or -acrylamide, styrene, vinylacetate orN-vinyllactam.

The block copolymer may be a simple A-B block copolymer, or may be anA-B-A or B-A-B block copolymer (where A is the zwitterionic block and Bis the ionic block). It may be a star-type polymer with more than twoarms of blocks A extending from a core block B or vice versa. It may bea comb type polymer in which the back bone is considered as block A andeach tine is a B block or vice versa. It may also be an A-B-C, A-C-B orB-A-C block copolymer, where C is a different type of block C blocksmay, for instance, comprise functional, e.g. cross-linking or ionicgroups, to allow for reactions of the copolymer, for instance in thenovel compositions. Crosslinking reactions especially of A-C-B typecopolymers, may confer useful stability on drug-containing micelles.Cross-linking may be covalent, or sometimes, electrostatic in nature.Crosslinking may involve addition of a separate reagent to linkfunctional groups, such as using a difunctional alkylating agent to linktwo amino groups.

The block copolymers preferably have controlled molecular weights. It ispreferable for each of the blocks to have molecular weight controlledwithin a narrow band, that is to have a narrow polydispersity. Thepolydispersity of molecular weight should, for instance, be less than2.0, more preferably less than 1.5, for instance in the range 1.1 to1.4.

The degree of polymerisation of an ionic block is in the range 5 to2000, preferably 10 to 500, more preferably 10 to 250. A zwitterionicblock has a degree of polymerisation in the range 2 to 1000, preferably5 to 250 more preferably 10 to 100. Generally the relative lengths ofthe ionic to zwitterionic blocks is in the range 1:5 to 10:1, preferably1:1 to 5:1.

It may be possible to synthesise the block copolymer by initialformation of a low polydispersity, low molecular weight initial blockusing control of initiator and chain transfer agent (which permanentlyterminates chain formation), with the initial block then beingderivatised to act as a suitable radical initiator in a subsequent blockforming step, by the technique described by Inoue et al J. Contr. Rel.1998, 51, 221-229. It may be possible to utilise commercially availablerelatively low molecular weight low polylispersity ionic polymers asstarting materials for a zwitterionic block-forming step, for instanceby derivatising the ionic polymer at one or both ends to generate aradical from which polymerisation of monomers including the zwitterionicmonomers may be initiated. Preferably, the polymerisation of at leastone of the blocks and preferably both the blocks is by controlledradical polymerisation for instance a living radical polymerisationprocess.

A living radical polymerisation process may be a group transfer radicalpolymerization, for instance in which an N—O, or other carbon-,sulphur-, and oxygen-centered radical group is transferred from aninitiator compound to a monomer. Preferably, however, the process is anatom transfer radical polymerisation process. Preferably such a processis used to form each block of the block copolymer.

In the atom or group transfer radical polymerisation process, theinitiator has a radically transferable atom or group, and the catalystcomprises a transition metal compound and a ligand, in which thetransition metal compound is capable of participating in a redox cyclewith the initiator and dormant polymer chain, and the ligand is eitherany N—, O—, P— or S—containing compound which can coordinate with thetransition metal atom in a σ-bond, or any carbon-containing compoundwhich can coordinate with the transition metal in a π-bond, such thatdirect bonds between the transition metal and growing polymer radicalsand not formed.

Preferably the radical initiator is of the general formula XIR²⁵R²⁶R²⁷C—X²  XIwhere:

-   X² is selected from the group consisting of Cl, Br, I, OR²⁸, SR²⁹,    SeR²⁹, OP(═O)R³⁰, OP(═O)(OR³⁰)₂, O—N(R³⁰)₂ and S—C(═S)N(R³⁰)₂, where    R²⁸ is alkyl of from 1 to 20 carbon atoms in which each of the    hydrogen atoms may be independently replaced by halide, R³⁰ is aryl    or a straight or branched C₁-C₂₀ alkyl group, and where an N(R³⁰)₂    group is present, the two R³⁰ groups may be joined to form a 5- or    6-membered heterocyclic ring; and

R²⁵, R²⁶ and R²⁷ are each independently selected from the groupconsisting of H, halogen, C₁-C₂₀ alkyl, C₃-C₈ cycloalkyl, C(═O)R³⁰,C(═O)NR³¹R³², COCl, OH, CN, C₂-C₂₀ alkenyl, C₂-C₂₀ alkenyl oxiranyl,glycidyl, aryl, heterocyclyl, aralkyl, aralkenyl, C₁-C₈ alkyl in whichfrom 1 to all of the hydrogen atoms are replaced with halogen, C₁-C₆alkyl substituted with from 1 to 3 substituents selected from the groupconsisting of C₁-C₄ alkoxy, aryl, heterocyclyl, C(═O)R³⁰, C(═O)NR³¹R³²,—CR²⁶R²⁷X², oxiranyl and glycidyl;

where R³⁰ is alkyl of from 1 to 20 carbon atoms, alkoxy of from 1 to 20carbon atoms, oligo(alkoxy) in which each alkoxy group has 1 to 3 carbonatoms, aryloxy or heterocyclyloxy any of which groups may havesubstituents selected from optionally substituted alkoxy, oligoalkoxy,amino (including mono- and di-alkyl amino and trialkyl ammonium, whichalkyl groups, in turn may have substituents selected from acyl,alkoxycarbonyl, alkenoxycarbonyl, aryl and hydroxy) and hydroxyl groups;and

R³¹ and R³² are independently H or alkyl of from 1 to 20 carbon atomswhich alkyl groups, in turn may have substituents selected from acyl,alkoxycarbonyl, alkenoxycarbonyl, aryl and hydroxy, or R³¹ and R³² maybe joined together to form an alkanediyl group of from 2 to 5 carbonatoms, thus forming a 3 to 6-membered ring;

such that not more than two of R²⁵, R²⁶ and R²⁷ are H.

In the initiator of the general formula V it is preferred that no morethan one of R²⁵, R²⁶ and R²⁷, and preferably none, is hydrogen. Suitablyat least one, and preferably both of R²⁵ and R²⁶ is methyl. R²⁷ issuitably a group CO—R³⁰ in which R³⁰ is preferably alkoxy of from 1 to20 carbon atoms, oligo(alkoxy) in which each alkoxy group has 1 to 3carbon atoms, aryloxy or heterocyclyloxy any of which groups may havesubstituents selected from optionally substituted alkoxy, oligoalkoxy,amino (including mono- and di-alkyl amino and trialkyl ammonium, whichalkyl groups, in turn may have substituents selected from acyl,alkoxycarbonyl, alkenoxycarbonyl, aryl and hydroxy) and hydroxyl groups.

Since any of R²⁵, R²⁶ and R²⁷ may comprise a substituent C²⁶R²⁷X², theinitiator may be di-, oligo- or poly-functional.

Selection of a suitable initiator is based on various considerations.Where the polymerisation is carried out in the liquid phase, in whichthe monomers are dissolved, it is preferable for the initiator to besoluble in that liquid phase. The initiator is thus selected for itssolubility characteristics according to the solvent system which in turnis selected according to the monomers being polymerised.

Water-soluble initiators include, for instance the reaction product ofmonomethoxy-capped oligo(ethylene oxide) with 2-bromoisobutyryl bromide(OEGBr), 4-bromo-α-toluic acid or ethyl 2-bromopropanoic acid or2-(N,N-dimethylamino)ethyl-2′-bromoisobutyrate.

The portion of the initiator —C—R²⁵R²⁶R²⁷ becomes joined to the firstmonomer of the growing polymer chain. The group X² becomes joined to theterminal unit of the polymer chain. Selection of a suitable initiator isdetermined in part by whether a terminal group having particularcharacteristics is required for subsequent functionality. The residue ofthe initiator at one or other end of the polymer may be reacted withbiologically active moieties, such as targetting groups. Alternativelythe initiator itself may comprise a group conferring useful targettingor other useful properties without further reaction.

In an atom or group radical transfer polymerisation process thetransition metal compound which comprises a component of the catalyst isM_(t) ^(n+)X³ _(n), where:

M_(t) ^(n+) may be selected from the group consisting of Cu¹⁺, Cu²⁺,Fe²⁺, Fe³⁺, Ru²⁺, Ru³⁺, Cr²⁺, Cr³⁺, Mo²⁺, Mo³⁺, W²⁺, W₃₊, Mn²⁺, Mn³⁺,Mn⁴⁺, Rh³⁺, Rh⁴⁺, Re²⁺, Re³⁺, Co⁺, Co²⁺, Co³⁺, V²⁺, V³⁺, Zn⁺, Zn²⁺,Ni²⁺, Ni³⁺, Au⁺, Au²⁺, Ag⁺ and Ag²⁺;

X³ is selected from the group consisting of halogen, C₁-C₆-alkoxy,(SO₄)_(1/2), (PO₄)_(1/3), (R³³PO₄)½, (R³³ ₂PO₄), triflate,hexafluorophosphate, methanesulphonate, arylsulphonate, CN and R³⁴CO₂,where R³³ is aryl or a straight or branched C₁₋₂₀ alkyl and R³⁴ is H ora straight or branched C₁-C₆ alkyl group which may be substituted from 1to 5 times with a halogen; and

n is the formal charge on the metal (0≦n≦7).

Preferably X³ is halide, most preferably chloride or bromide.Particularly suitable transition metal compounds are based on copper orruthenium, for instance CuCl, CuBr or RuCl₂.

In the catalyst, the ligand is preferably selected from the groupconsisting of:

a) compounds of the formulas:R³⁵—Z—R³⁶andR³⁵—Z—(R³⁷—Z)_(m)-R³⁶where:

R³⁵ and R³⁶ are independently selected from the group consisting of H,C₁-C₂₀ alkyl, aryl, heterocyclyl and C₁-C₆ alkoxy, C₁-C₄ dialkylamino,C(═O)R³⁶, and A¹¹C(═O)R⁴⁰, where A¹¹ may be NR⁴¹ or O; R³⁸ is alkyl offrom 1 to 20 carbon atoms, aryloxy or heterocyclyloxy; R⁴⁰ is H,straight or branched C₁-C₂₀ alkyl or aryl and R⁴¹ is hydrogen, straightor branched; C₁₋₂₀-alkyl or aryl; or R³⁵ and R³⁶ may be joined to form,together with Z, a saturated or unsaturated ring;

Z is O, S, NR⁴² or PR⁴², where R⁴² is selected from the same group asR³⁵ and R³⁶, and where Z is PR⁴², R⁴² can also C₁-C₂₀ alkoxy or Z may bea bond, CH₂ or a fused ring, where one or both of R³⁵ and R³⁶ isheterocyclyl,

-   -   each R³⁷ is independently a divalent group selected from the        group consisting of C₁-C₈ cycloalkanediyl, C₁-C₈        cycloalkenediyl, arenediyl and heterocyclylene where the        covalent bonds to each Z are at vicinal positions or R³⁷ may be        joined to one or both of R³⁵ and R³⁶ to formulate a heterocyclic        ring system; and

m is from 1 to 6;

b) CO;

c) porphyrins and porphycenes, which may be substituted with from 1 to 6halogen atoms, C₁₋₆ alkyl groups, C₁₋₆alkoxy groups, C₁₋₆alkoxycarbonyl, aryl groups, heterocyclyl groups, and C₁₋₆ alkyl groupsfurther substituted with from 1 to 3 halogens;

d) compounds of the formula R⁴³R⁴⁴C(C(═O)R⁴⁵)₂, where R⁴⁵ is C₁₋₂₀alkyl, C₁₋₂₀ alkoxy, aryloxy or heterocyclyloxy; and each of R⁴³ and R⁴⁴is independently selected from the group consisting of H, halogen, C₁₋₂₀alkyl, aryl and heterocyclyl, and R⁴³ and R⁴⁴ may be joined to form aC₁₋₈ cycloalkyl ring or a hydrogenated aromatic or heterocyclic ring, ofwhich the ring atoms may be further substituted with 1 to 5 C₁₋₆ alkylgroups, C₁₋₆ alkoxy groups, halogen atoms, aryl groups, or combinationsthereof; and

e) arenes and cyclopentadienyl ligands, where said cyclopentadienylligand may be substituted with from one to five methyl groups, or may belinked through and ethylene or propylene chain to a secondcyclopentadienyl ligand.

Selection of a suitable ligand is, for instance, based upon thesolubility characteristics and/or the separability of the catalyst fromthe product polymer mixture. Generally it is desired for the catalyst tobe soluble in a liquid reaction mixture, although under somecircumstances it may be possible to immobilise the catalyst, forinstance an a porous substrate. For the preferred process, which iscarried out in the liquid phase, the ligand is soluble in a liquidphase. The ligand is generally a nitrogen containing ligand. Thepreferred ligand may be a compound including a pyridyl group, such asbipyridine, a compound including a pyridyl group and an imino moietysuch as:

where R⁴⁶ is a suitable alkyl group, the substituent being variable andadaptable to confer desired solubility characteristics,triphenylphosphine or 1,1,4,7,10,10-hexamethyl-triethylene tetramine.

Such ligands are usefully used in combination with copper chloride,copper bromide and ruthenium chloride transition metal compounds as partof the catalyst.

A living radical polymerisation process is preferably carried out toachieve a degree of polymerisation in the or each block in the range 5to 500. Preferably the degree of polymerisation is in the range 10 to100, more preferably in the range 10 to 50. In the preferred group oratom transfer radical polymerisation technique, the degree ofpolymerisation is directly related to the initial ratios of initiator tomonomer. Preferably the ratio is in the range 1:(5 to 500), morepreferably in the range of 1:(10 to 100), most preferably in the range1:(10 to 50).

The ratio of metal compound and ligand in the catalyst should beapproximately stoichiometric, based on the ratios of the components whenthe metal ion is fully complexed. The ratio should preferably be in therange 1:(0.5 to 2) more preferably in the range 1:(0.8:1.25). Preferablythe range is about 1:1.

In the living radical polymerisation process, the catalyst may be usedin amounts such that a molar equivalent quantity as compared to thelevel of initiator is present. However, since catalyst is not consumedin the reaction, it is generally not essential to include levels ofcatalyst as high as of initiator. The ratio of catalyst (based ontransition metal compound) to initiator is preferably in the range 1:(1to 50), more preferably in the range 1:(1 to 10).

Whilst the polymerisation reaction may be carried out in the gaseousphase, it is more preferably carried out in the liquid phase. Thereaction may be heterogeneous, that is comprising a solid and a liquidphase, but is more preferably homogeneous. Preferably the polymerisationis carried out in a single liquid phase. Where the monomer is liquid, itis sometimes unnecessary to include a non-polymerisable solvent. Moreoften, however, the polymerisation takes place in the presence of anon-polymerisable solvent. The solvent should be selected having regardto the nature of the zwitterionic monomer and any comonomer, forinstance for its suitability for providing a common solution containingboth monomers. The solvent may comprise a single compound or a mixtureof compounds.

It has been found that, especially where the zwitterionic monomer isMPC, that it is desirable to include water in the polymerisationmixture. Preferably water should be present in an amount in the range 10to 100% by weight based on the weight of ethylenically unsaturatedmonomer. Preferably the total non-polymerisable solvent comprised 1 to500% by weight based on the weight of ethylenically unsaturated monomer.It has been found that the zwitterionic monomer and water should be incontact with each other for as short a period as possible prior tocontact with the initiator and catalyst. It may be desirable thereforefor all the components of the polymerisation other than the zwitterionicmonomer to be premixed and for the zwitterionic monomer to be added tothe premix as the last additive.

It is often desired to copolymerise MPC or other zwitterionic monomerwith a comonomer which is insoluble in water. In such circumstances, asolvent or cosolvent (in conjunction with water) is included to confersolubility on both MPC and the more hydrophobic monomer. Suitableorganic solvents are ethers, esters and, most preferably, alcohols.Especially where a mixture of organic solvent and water is to used,suitable alcohols are C₁₋₄-alkanols. Methanol is found to beparticularly suitable in the polymerisation process of the invention.

The process may be carried out at raised temperature, for instance up to60 to 80° C. However it has been found that the process proceedssufficiently fast at ambient temperature.

The living radical polymerisation process has been found to provideblocks of zwitterionic monomers having a polydispersity (of molecularweight) of less than 1.5, as judged by gel permeation chromatography.Polydispersities in the range 1.2 to 1.4 for the or each block arepreferred.

In the composition the relative amounts of biologically active compoundand of polymer may be about stoichiometric in terms of the counterionicgroups. Alternatively there may be an excess of one charge over theother for instance up to 5 or 10 times excess. The level may depend onstability factors or on interactions of the components of thecomposition with biological systems. For instance, Rungsardthong, et al,op. cit., show that the level of excess cationic polymer over DNA mayaffect transfection levels. Appropriate levels of the biologicallyactive compound and polymer may be determined by experimentation. Theparticles may be analysed for their ζ potential. This techniquedetermines the presence of overall charge on the surface of particles.Stability and activity may be determined by available assays. It hasbeen found that the presence of zwitterionic groups in the aqueouscomposition stabilises the dispersed particles, without requiringaddition of stabilisers, or using an excess of drug or polymer. Thecompositions may additionally contain buffers or other salts orpH-modifying components, stabilisers etc.

Further probes into the particles in the composition may be by dynamiclight scattering investigations, which may give a value for averageaggregate diameters. Small angle neutron scattering may also be used toprovide structural details inside the particles, as may electronmicroscopy, for instance transmission electron microscopy (TEM).

According to a further aspect of the invention there is provided aprocess for forming the novel composition in which an aqueous dispersionof a block copolymer having an overall ionic charge and comprising atleast one block which has pendant zwitterionic groups and at least oneblock which comprise ionic groups to confer said overall ionic charge,is contacted with a biologically active compound having a chargeopposite that of the block copolymer, to form an aqueous suspension ofblock copolymer and associated active.

In the process the dispersion of copolymer may be a solution including acolloidal solution or a suspension. The copolymer may be in the form ofmicelles, for instance. The pH is selected so as to ensure that thecounterionically charged groups in polymer and ionic groups inbiologically active attract each other electrostatically.

Preferably the active is pre-dissolved in water before being contactedwith the copolymer dispersion, although direct dissolution into theblock copolymer dispersion may be possible. We have found that simplephysical mixing of the aqueous solutions results in suspended particlesof a suitable size for administration for therapeutic purposes.

It is believed that the compositions will be particularly useful forallowing gene or ODN delivery into cells. Thus the compositions may beuseful for administering to the patients in need of therapy by thebiologically active molecule. The compositions may thus be suitable foradministration IV, IP or IM for instance. The effect of the compositionson intracellular delivery may be illustrated using in vitro testsystems. For instance delivery of genes into cells may be determined byusing, as the biologically active molecule, a plasmid encoding a modelgene, the product of which may be observed. Suitable vectors areavailable encoding luciferase and/or β-galactosidase. Such tests may becarried out in conjunction with cell proliferation assays usingtritium-labelled thymidine and using as positive controls known cationicpolymer delivery systems such as poly-L-lysine, or PEO-PEI blockcopolymers.

Cytotoxicity determinations may be conducted. Such tests may, forinstance, be useful to determine the base toxicity of the polymersthemselves. Alternatively, where the drug to be delivered is intended tobe cytotoxic, a cytotoxicity test may reveal the success of the drugdelivery system. Stability of the compositions may be investigated, forinstance by contacting the compositions with pH modifiers, salts and/orserum and determining physical and biologically stability. Resistance todegradation by enzymic reactions, such as attack of nucleic acid bynucleases is relevant to the utility of the invention in formulatingnucleic acids, as is the effect on ethidium bromide intercalation.

The following examples illustrate the invention.

EXAMPLE 1

A-B block copolymers were formed by an atom transfer polymerisation withMPC being homopolymerised in a first block forming step using anoligo(ethylene glycol) initiator as described by Ashford E. J. et al inChem. Commun. 1999, 1285 (the reaction product of monomethoxy-cappedoligo(ethylene glycol) and 2-bromoisobutyryl bromide) in the presence ofbipyridine ligand and copper (I) bromide catalyst. DEA(diethylaminoethyl methacrylate) was polymerised in a second blockforming step. The degree of polymerisation for each block is indicatedin Table 1 showing the results.

The reaction conditions were [MPC]=2.02M (6.0 g in 10 ml methanol),[MPC]: [OEG-Br]:[CuBr]:[bipy]=(30 or 20 as shown in Table 1):1:1:2,T=20° C.; MPC was polymerised first in all cases followed by additionand polymerisation of an appropriate amount of neat DEA. Almost completemonomer conversion was achieved after the time indicated in Table 1 forthe diblock, as indicated by ¹H NMR spectroscopy (no residual vinyldouble bonds). The reaction mixture was diluted with methanol and passedthrough a silica column to remove residual ATRP catalyst. After solventevaporation, the products were dried under vacuum at room temperature.

TABLE 1 Data of the polymerization of MPC - DEAEMA diblock copolymers inmethanol Time for >99% Conversion MPC In MPC MPC Mn (AGPC) Mw/Mncopolymer [Amine] HOMO Diblock MPC(a) MPC(b) MPC MPC Ex # Comonomer (mol%) TargetDp (M) (mins) (h) HOMO Diblock HOMO Diblock 1 DEA 50 20:20 1.35180 20  6200 14000 1.15 1.22 2 DEA 33 10:20 1.35 180 21  3500 11000 1.171.29 3 DEA 50 30:30 2.02 130 20 10000 21000 1.18 1.30 4 DEA 33 30:604.04 130 22 11000 31000 1.19 1.29 5 DEA 23  30:100 6.73 130 23 1100043000 1.19 1.28 DEA = diethylaminoethylmethacrylate AGPC = aqueous gelpermeation chromatography

The MPC-DEA block copolymers were dissolved in McIlvaines buffer at aconcentration of 1 mM and at pH 4. The pH was then adjusted upwards withNaOH to pH 8, and 10.8, so the micelles would form. A series of halfdilutions were then prepared from the micellised polymers, usingMcIlvaines buffer of the same pH as the polymer solution.

To demonstrate the polymer shift from unimer to micelle state inresponse to pH increase, a control of polymer at pH 4 was carried usingthe same technique and conditions as that used for the pH8 and pH10.8samples using the 30:60 MPC:DEA block copolymer. The polymer solutionsat pH4 and pH8 were also analysed using photon correlation spectroscopy(PCS) to measure the hydrodynamic diameter of the particles based on theintensity of scattered light, and calculated using the Stokes-Einsteinequation, as described in ISO13321 British Standards Institution. 1997,BS3046: Part 8: 1997: ISO 13321: “Photon correlation spectroscopy”, inMethods for determination of is particle size distribution, BSIpublications, Chiswick, UK, p 1-21, with subsequent analysis anddetermination of intensity size distributions using the CONTINalgorithm. Measurement was carried out using a Malvern Zetasizer 3000HS,using a 10 mW He—Ne laser, with a wavelength of 633 nm, and a highsensitivity avalanche photodiode detector fixed at a 90 degree angle tothe laser, at a temperature of 25° C. Samples were sonicated for 5minutes and filtered through a 0.2 micron filter prior to measurement,to remove any aggregation and possible dust contamination.

In FIG. 1 the shift from unimer to micelle in response to increased pHcan be seen. At pH4 (grey shaded curve) only unimers with a meandiameter of 11.3 nm are present, however when the pH is raised to pH8(black shaded curve) there is a clear increase in mean diameter from11.3 nm up to 37.5 nm, indicating the unimers have undergone micellarself assembly in response to increased pH.

EXAMPLE 2 Generic Block Copolymer Preparation by Sequential MonomerAddition

MPC was polymerized first in 10 ml methanol, using[MPC]:[OEGBr]:[CuBr]:[bpy]=X:1:1:2 (where X is the number of moles ofMPC used to achieve the desired target degree of polymerization) under anitrogen atmosphere at 20° C. After 2.0 h, the MPC conversion wasgreater than 99%, and the MPC homopolymer obtained had a lowpolydispersity (Mw/Mn=1.09 with Mn=10,000 vs. poly(ethylene oxide)standards. Then the appropriate amount of the comonomer2-dimethylaminoethyl methacrylate (DMA) was added to this reactionsolution, to give a particular target degree of polymerization for thesecond block. After 40 h, ¹H NMR studies indicated that both monomershad been consumed. The reaction solution was passed through a silica gelcolumn to remove the spent ATRP catalyst, which resulted in the loss ofaround 10% copolymer due to adsorption onto the silica. After solventevaporation, the solid copolymer was washed with an excess of a suitablesolvent to remove any traces of residual comonomer, redissolved in waterand then freeze-dried overnight. The molecular weight and polydispersityof the resulting polymers was determined by aqueous GPC usingpoly(2-vinylpyridine) standards. Table 2 summarises data for a series ofMPC-DMA diblock copolymers.

TABLE 2 Time for >95% Conversion^(a) Target Homo Diblock Mn (GPC)^(b)Mw/Mn^(b) Cu^(c)/ Composition (h) (h) Homo Diblock Homo Diblock ppm 1MPC₃₀-DMA₁₀₀ 2.0 48 10,000 46,000 1.17 1.32 1.7 2 MPC₃₀-DMA₆₀ 2.0 2410,000 34,000 1.15 1.28 1.5 3 MPC₃₀-DMA₄₀ 2.0 20  9,000 25,000 1.15 1.261.2 4 MPC₃₀-DMA₃₀ 2.0 15 10,000 22,000 1.19 1.27 2.0 5 MPC₃₀-DMA₂₀ 2.010 10,000 18,000 1.18 1.22 0.8 6 MPC₃₀-DMA₁₀ 2.0  8 10,000 15,000 1.171.21 0.6 7 MPC₆₀-DMA₄₀ 1.6 24 14,000 22,000 1.18 1.28 0.5 8 MPC₄₀-DMA₄₀1.3 24 11,000 19,000 1.15 1.27 0.9 9 MPC₂₀-DMA₄₀ 1.1 20  6,000 15,0001.10 1.25 0.4 10 MPC₁₀-DMA₄₀ 1.0 18  3,000 30,000 1.07 1.23 0.4 ^(a)Asdetermined by ¹H NMR spectroscopy. ^(b)As determined by aqueous GPC atpH 2 (0.5M acetic acid + 0.03M sodium acetate) usingpoly(2-vinylpyridine) calibration standards. ^(c)As determined byinductively-coupled plasma atomic emission spectroscopy [ICP-AES].

EXAMPLE 3 Agarose Gel Electrophoresis

The interaction between plasmid DNA (Calf thymus DNA, Sigma UK) andpolymers was investigated by electrophoresis on agarose gel (PromegaCorporation, Madison, USA). Complexes were prepared corresponding tomonomer:nucleotide molar ratio (mole/mole) in terms of DMA polymerrepeating units: DNA nucleotides. Complexes at monomer:nucleotide molarratio of 0.2:1, 0.5:1, 1:1, 1.2:1, 1.4;1, 1.8:1, 2:1, 5:1 and 10:1 wereplaced in the wells of the 0.6% agarose gel containing ethidium bromide(0.6 μg/ml). Solution containing 1 mg plasmid DNA was used. Controls forfree DNA and free polymers were also applied on the gel. Gel runningbuffer was 40 mM Tris acetate (pH 7.4) and 1 mM EDTA. The gel was run at70 V for one hour, after which the DNA was visualized on a UVtransilluminator. Polymer was visualized by staining the gel with dye(Coomassie blue in 10% acetic acid and 10% ethanol) and subsequentwashing with the destaining solution (same as the staining solutionwithout the dye).

Electrophoretic movement of the DNA molecule within the gel is inhibitedby complexation with the MPC polycation, in this case MPC20-DMA20. Lanes1 and 11 represent the controls of free plasmid DNA and free polymerrespectively. Lanes 2-10 represent the polycation monomer to DNA baseratios of 0.2, 0.5, 1, 1.2, 1.4, 1.8, 2, 5 and 10. Retardation ofcomplexed/condensed DNA in the application well can be seen in theFigures. Visualisation of the gel in FIG. 2( a) was achieved by DNAintercalating fluoresce probe (ethydium bromide) incorporation in thegel. The presence of migrating DNA in lanes 2-6 indicate that there isinsufficient polymer present to completely condense all of the DNA thatis present into an aggregate. Eventual inhibition of the DNA migratingband in FIG. 2( a) occurred in lane 7, which was equivalent to ratio1.4. The free polymer bands in FIG. 2( b) indicated the presence ofexcess of the polymer at certain ratios which is not associated with thecondensates. Likewise, appearance of free polymer bands appear at Lane 7and greater, again at a ratio of 1.4. Thus, the MPC-DMA diblock polymersystem is capable of condensing with DNA, and for the MPC20-DMA20 systemstudied here, a ratio of 1:1.4 for DNA:polymer was required to producean optimum aggregate.

EXAMPLE 4 DNA Binding Affinity by Ethidium Bromide Displacement

An assessment of the MPC-polycations' ability to form complexes with DNAwas made by measuring changes in the fluorescence of ethidiumbromide-DNA complexes. Loss of ethidium bromide fluorescence is thoughtto result from polyelectrolyte binding leading to a DNA complexation,resulting in expulsion of intercalated ethidium bromide molecules. Thelevel of fluorescence reduction is related to the affinity of MPCpolycation-DNA binding. Ethidium bromide (2 μg, 1 mg/ml) was added to 10fold diluted phosphate buffer saline (1000 μl) in cuvettes and mixed bygentle agitation Fluorescence was recorded in triplicate at I_(ex) 560and I_(em) 605 nm in a Hitachi F-4500 fluorescence spectrophotometer.Calf thymus DNA (10 μg) was added and fluorescence measured again. Analiquot of polymer was then titrated into the solution to a calculatedmonomer nucleotide molar ratio. Samples were mixed gently and readingswere taken after 1-2 min. Duplicate samples were used for eachmeasurement Complex formation in the presence of salt was assessed byvarying NaCl concentration i.e. 10, 25, 50, 1000, 2000 mM in a ten folddilution of phosphate buffer saline (1000 μl). Control readings weretaken using PEG 4000 Da solution.

The relative fluorescence was calculated as below:

${\%\mspace{11mu}{Relative}\mspace{14mu}{Fluor}^{\prime}{ce}} = {\frac{{{Fluor}^{\prime}{{ce}({obs})}} - {{Fluor}^{\prime}{{ce}({EtBr})}}}{{Fluor}^{\prime}{{ce}\left( {{DNA} + {EtBr} - {{Fluor}^{\prime}{{ce}({EtBr})}}} \right.}} \times 100}$

-   Fluor'ce (obs)=Fluor'ce of DNA+Ethidium Bromide+polymer-   Fluor'ce (EtBr)=Fluor'ce of Ethidium Bromide alone-   Fluor'ce (DNA+EtBr) Fluor'ce of DNA+Ethidium Bromide

FIG. 3 compares the ethidium bromide displacement of a number ofpolymers, including

(i) the MPC30-DMA30 diblock made in example 2,

(ii) DMA homopolymer, having an average MW6050 and polyolispersity1.8“DMAEMA”.

(iii) polyvinylpyrrolidone (Aldrich) with average MW29KD (PVP).

(iv) DMA-PEG linear diblock copolymer having an average MW9750, a DMAcontent of 40 mol % and polydispersity 1.25 (synthesised by anoxyanionic polymerisation as described in Varnvakaki, M et al inMacromolecules 32 (1999) 2088-2090). It is similar to one of thepolymers tested in Rungsardthong et al., op cit.

(v) two diblock copolymers formed by polymerising a first block of DMAand a second block of methoxy-capped oligo (ethylene glycol)methacrylate having an average number of ethylene glycol repeat units of7.5 as described in more detail by Bailey, L, PLD thesis, University ofSussex 2000. The polymers were similar to those tested by Rungsardthonget al., op cit. DMA-OEGMA15 has an average MW of 11990, a polydispersityof 1.09 and a DMA content of 66 mol %. DMA-OEGMA7 has an average MW of9630, a polydispersity of 1.12 and a DMA content of 84 mol %.

A reduction in the relative fluorescence indicates ability of thepolymer to interact with DNA displacing the probe. Clearly, the MPCdiblock and DMA homopolymer have the ability to condense the DNA, as areduction in the relative fluorescence indicates ability of the polymerto interact with the DNA and thus displacing the probe. The control PVPpolymer did not have this capability. The DMA-MPC diblock required ahigher ratio of cationic monomer polymer to DNA in order to condensecompared to DMA monopolymer or any of the poly(ethylene-glycol)-groupcontaining polymers. This may be a consequence of the MPC block of thepolymer affecting the condensation process.

FIG. 4 shows the ethidium bromide displacement ability for a series ofDMAx-MPC30 copolymers (where x=10, 20, 30, 40, 100) at different monomernucleotide ratios. There was a rapid decrease in the relativefluorescence while increasing the ratio from 0.2 to 1.0. Between ratios1.0 to 5.0, a gradual decrease in fluorescence was observed withincreasing ratio. At ratios higher than 5.0, a plateau was reached. Theminimal level of fluorescence reached ranged from 25-40%. The DMA₁₀MPC₃₀ copolymer showed the least decrease in fluorescence initially,indicating that it was least able to bind with DNA. The DMA₄₀MPC₃₀showed a lesser extent of DNA binding when compared with other polymers.For all other DMA-co-MPC polymers, the displacement profile was similarto the DMA homopolymer. Thus, the presence of the hydrophilic MPC doesnot appear to hinder the interaction between DNA and DMA portion of thecopolymers. Indeed, the MPC homopolymer showed some ability to interactwith the DNA, but to a much lesser extent than the copolymers. FIG. 5shows the effect of varying the MPC block length for DMA40MPCX blockcopolymers; this had little impact on the ability to interact with theDNA for MPC block lengths 10 to 50 monomer units long (i.e X=10-50).

EXAMPLE 5 Scattering Intensity and Particle Size Characterisation

The scattering intensity of polymer/DNA complexes was measured by aMalvern S4700 PCS system (Malvern Instrument, Malvern UK). The study wasundertaken by the titration of polymers into 10 μg of calf thymus DNAprepared in ten fold diluted phosphate buffer saline (500 μl). Thesamples were then mixed by gentle agitation after the aliquot of polymerwas added. Measurements were made at different monomer:nucleotide molarratios at 25° C. using a 40.7 mW laser and a scattering angle of 90°.The scattering intensity of each sample was obtained as the mean of 10determinations. Data presented are the mean of a minimum of 2 replicatetitrations. The diameter of polymer/DNA complexes was also measured by aMalvern S4700 PCS system. Individually prepared complexes of pCT0129LDNA (10 μg) (pCT0129LDNA, a 4.3-kb expression vector containing thechloramphenicol actyltransferase reporter gene was used as obtained fromGene Medicine Inc (Texas, USA)) in ten fold diluted phosphate buffersaline (500 μl) and addition of a aliquot polymer were measured atdifferent monomer nucleotide molar ratios. The measurements wereperformed at 25° C., 40.7 mW laser and a scattering angle 90°. Theparticle size of each sample was obtained by using CONTIN analysis asthe mean hydrodynamic diameter±standard deviation of six determinationsincluding scattering intensity and polydispersity.

Using PCS to monitor complexation, as the monomer:nucleotide molarratios exceeded 1.0, the size of complexes formed by the DMA homopolymerincreased significantly to above 1 μm, indicating that agglomeration ofparticles occurred (FIG. 6). At ratio 1.0, the complexes become neutraland there is insufficient electrostatic repulsion to stabilise thecomplexes and prevent aggregation.

Table 3 shows the particles size and scattering intensity for MPC30 DMAXdiblock copolymers. Table 4 show the particle size and scatteringintensity data for MPCX-DMA 40 diblocks.

The DMA MPC polymers complexes exhibited a different behaviour.Generally, the complexes formed by MPC-based copolymers were colloidallystable, with an average size of 150 nm. The complexes remained small anddiscrete as the monomer nucleotide molar ratios exceeded 1.0.Incorporation of hydrophilic MPC moiety to cationic polymer can thusprovide steric stabilization and prevent aggregation. Complexes formedwith DMA₁₀MPC₃₀ were found to have a larger size (>200 nm) at highmonomer nucleotide molar ratio. It was unlikely that agglomerationoccurred as the complexes size was still significantly lower than thatof DMA homopolymer. One of the possible explanations is thatdissociation of complexes could have happened.

For complexes with a smaller proportion of DMA (DMA₁₀MPC₃₀ andDMA₂₀MPC₃₀), the scattering intensity was lower compared with those witha higher proportion of DMA, indicating that more complexes were formed(FIG. 7). The amount of complexes formed correlates to the proportion ofDMA, which is responsible for the binding of DNA. It was also found thatincreasing the proportion of DMA results in uniform complex size, as thepolydispersity decreased with longer DMA chain. For DMA₁₀₀MPC₃₀, thescattering intensity was significantly higher than other MPC-basedcopolymer although the complexes see was about the same, suggesting thateither the complexes formed were very condensed or they were relativelyinsoluble.

TABLE 3 Paricle size ± SD (nm) (Polydispersity ± SD) Monomer:NucleotideScattering Intensity ± SD molar ratio DMA₁₀MPC₃₀ DMA₃₀MPC₃₀ DMA₁₀₀MPC₃₀0.2 585.7 ± 50.1 (1.0 ± 0) 275.1 ± 38.9 (0.884 ± 0.099)  131.2 ± 12.1(0.339 ± 0.164)  38.4 ± 2.8 184.0 ± 16.2  556.8 ± 261.4 0.5 151.0 ± 23.3(0.645 ± 0.11) 215.0 ± 0.4 (0.349 ± 0.011)  149.1 ± 13.1 (0.238 ± 0.037) 67.5 ± 3.7 331.6 ± 10.0 1214.7 ± 186.1 0.7 101.5 ± 16.6 (0.748 ± 0.437)180.5 ± 0.9 (0.275 ± 0.008)  147.9 ± 4.9 (0.172 ± 0.076) 186.4 ± 9.3854.5 ± 2.3 1654.0 ± 168.9 0.8 157.2 ± 8.7 (0.514 + 0.030) 197.4 ± 1.6(0.284 ± 0.006)  180.7 ± 19.1 (0.260 ± 0.029) 349.0 ± 16.8 651.8 ± 5.81526.6 ± 59.1 0.9 149.0 ± 7.2 (0.522 ± 0.032) 167.1 ± 1.3 (0.362 ±0.015)  120.3 ± 0.3 (0.120 ± 0.029) 371.5 ± 15.4 407.2 ± 6.7 1122.0 ±194.8 1 294.4 ± 87.5 (0.877 ± 0.189) 170.6 ± 2.7 0.325 ± 0.011  130.1 ±10.2 (0.170 ± 0.029) 508.6 ± 71.7 474.0 ± 4.5 1598.7 ± 658.7 2 202.4 ±60.3 (0.700 ± 0.254) 113.4 ± 2.1 (0.326 ± 0.047)  141.0 ± 13.2 (0.220 ±0.026) 418.0 ± 53.5 491.4 ± 13.9 1467.9 ± 61.9 3 126.3 ± 8.1 (0.473 ±0.078)  106.9 ± 2.6 (0.189 ± 0.020) 442.4 ± 14.4  813.1 ± 106.0 5 394.5± 159.3 (0.686 ± 0.236) 148.3 ± 1.8 (0.418 ± 0.018)  125.2 ± 9.0 (0.211± 0.008) 385.8 ± 45.6 550.8 ± 45.7 1052.1 ± 103.9 10 109.2 ± 6.9 (1.0 ±0) 169.9 ± 20.5 (0.525 ± 0.114)  120.2 ± 12.2 (0.207 ± 0.005) 365.4 ±11.7 539.7 ± 23.7 1004.3 ± 90.1

TABLE 4 Particle size ± SD (nm) (Polydispersity ± SD) Monomer:NucleotideScattering intensity ± SD molar ratio DMA Homopolymer DMA40MPC10DMA40MPC30 0.2:1  226.0 ± 1.7 (0.403 ± 0.013) 245.5 ± 27.2 (0.532 ±0.194) 174.9 ± 8.7 (0.499 ± 0.017)  292.3 ± 2.0  53.8 ± 0.6  70.4 ± 11.60.3:1  177.5 ± 8.1 (0.39 ± 0.064) 244.6 ± 17.1 (0.585 ± 0.049)  452.7 ±11.3  92.2 ± 7.0 0.5:1  167.5 ± 2.9 (0.376 ± 0.037) 203.3 ± 8.7 (0.409 ±0.044) 249.9 ± 18.5 (0.679 ± 0.051)  527.2 ± 6.5 240.5 ± 6.1 130.0 ± 6.40.6:1  154.4 ± 2.1 (0.322 ± 0.024) 259.9 ± 21.2 (0.337 ± 0.076) 137.5 ±2.6 (0.345 ± 0.019)  738.9 ± 11.0 263.1 ± 11.2 107.5 ± 5.6 0.7:1  195.6± 5.1 (0.304 ± 0.013)   290 ± 11.4 (0.258 ± 0.041) 161.5 ± 4.6 (0.419 ±0.028) 1910.8 ± 23.1 192.3 ± 4.8 156.2 ± 9.0 0.8:1  540.4 ± 140.2 (0.459± 0.067) 320.9 ± 6.0 (0.326 ± 0.031) 148.5 ± 2.4 (0.344 ± 0.007) 2658.5± 24.7 145.8 ± 8.6 153.7 ± 14.0 0.9:1 1243.3 ± 107.4 (0.416 ± 0.031)383.7 ± 16.1 (0.429 ± 0.014) 154.5 ± 2.1 (0.373 ± 0.01) 2225.5 ± 69.4118.0 ± 1.7 172.5 ± 3.3 1.0:1 1704.0 ± 99.3 (0.691 ± 0.103) 411.7 ± 21.2(0.482 ± 0.083) 141.4 ± 1.7 (0.223 ± 0.005) 2686.8 ± 69.0 105.7 ± 3.8288.1 ± 5.5 2.0:1 1411.6 ± 172.1 (0.5 ± 0.036) 443.6 ± 22.7 (0.497 ±0.047) 127.2 ± 0.7 (0.299 ± 0.009) 2714.4 ± 52.7 102.3 ± 5.0 415.6 ± 3.45.0:1 1436.7 ± 178.5 (0.568 ± 0.049) 527.8 ± 21.7 (0.433 ± 0.016) 123.3± 0.009 (0.312 ± 0.009) 2686.9 ± 39.7 102.4 ± 4.9 358.6 ± 3.7

1. A composition comprising a block copolymer having an overall ioniccharge and associated with the polymer a biologically active compoundhaving a charge opposite that of the polymer and is characterised inthat block copolymer comprises at least one zwitterionic block which haspendant zwitterionic groups and at least one ionic block which compriseionic groups to confer said overall ionic charge, wherein thebiologically active compound is anionic.
 2. A composition according toclaim 1 in which the active compound is a nucleic acid.
 3. A compositionaccording to claim 2 in which the nucleic acid is selected from thegroup consisting of oligo nucleotides, having 5 to 80 bases, singlestranded RNA, single stranded DNA and double stranded DNA.
 4. Acomposition according to claim 1 in which the biologically activecompound is an anionic drug.
 5. A composition according to claim 1 inwhich the biologically active compound and polymer are associated withone another in the form of particles having an average diameter lessthan 200 μm.
 6. A composition according to claim 5 which is an aqueouscomposition in which the particles are suspended.
 7. A compositionaccording to claim 1 in which the zwitterionic block is formed fromethylenically unsaturated monomers including a zwitterionic monomerhaving the general formulaYBX  I in which Y is an ethylenically unsaturated group selected fromthe group consisting of H₂C═CR—CO-A-, H₂C═CR—C₆H₄-A¹-, H₂C═CR—CH₂A²,R²O—CO—CR═CR—CO—O, RCH═CH—CO—O—, RCH═C(COOR²)CH₂—CO—O,

A is —O— or NR¹; A^(l) is selected from the group consisting of a bond,(CH₂)_(l)A² and (CH₂)_(l) SO₃— in which l is 1 to 12; A² is selectedfrom the group consisting of a bond, —O—, O—CO—, CO—O, CO—NR¹—, —NR¹—CO,O—CO—NR¹—, and NR¹—CO—O—; R is hydrogen or C₁₋₄ alkyl; R¹ is hydrogen,C₁₋₄₋ alkyl or BX; R² is hydrogen or C₁₋₄ alkyl; B is selected from thegroup consisting of a bond, straight and branched alkanediyl groups,alkylene oxaalkylene groups, and alkylene (oligooxalkylene) groups,optionally containing one or more fluorine substituents; and X is azwitterionic group.
 8. A composition according to claim 7 in which Xcomprises a cation selected from the group consisting of ammonium,phosphonium and sulphonium groups and an anion selected from the groupconsisting of phosphate and phosphonate ester groups.
 9. A compositionaccording to claim 8 in which X has the general formula II

in which the moieties A³ and A⁴, which are the same or different, are—O—, —S—, —NH— or a valence bond and W⁺is a group comprising anammonium, phosphonium or sulphonium cationic group and a group linkingthe anionic and cationic moieties which is a C₁₋₁₂ alkanediyl group. 10.A composition according to claim 9 in which W⁺is a group of formula—W¹—N⁺R³ ₃, —W¹—P⁺R⁴ ₃, —W¹—S⁺R⁴ ₂ or —W¹-Het⁺ in which: W¹ is selectedfrom the group consisting of alkanediyl of 2-6 carbon atoms optionallycontaining one or more ethylenically unsaturated double or triple bonds,disubstituted-aryl (arylene), alkylene arylene, arylene alkylene, andalkylene aryl alkylene, cycloalkanediyl, alkylene cycloalkyl, cycloalkylalkylene or alkylene cycloalkyl alkylene, which group W¹ optionallycontains one or more fluorine substituents and/or one or more functionalgroups; and either the groups R³ are the same or different and each isselected from the group consisting of hydrogen, alkyl of 1 to 4 carbonatoms, and aryl or two of the groups R³ together with the nitrogen atomto which they are attached form an aliphatic heterocyclic ringcontaining from 5 to 7 atoms, or the three groups R³ together with thenitrogen atom to which they are attached as heteroaromatic ring having 5to 7 atoms, either of which rings may be fused with another saturated orunsaturated ring to form a fused ring structure containing from 5 to 7atoms in each ring, and optionally one or more of the groups R³ issubstituted by a hydrophilic functional group, and the groups R⁴ are thesame or different and each is R³ or a group OR³, where R³ is as definedabove; and Het is an aromatic nitrogen-, phosphorus- orsulphur-containing ring.
 11. A composition according to claim 7 in whichX has the general formula III

where the groups R⁵ are the same or different and each is hydrogen orC₁₋₄ alkyl, and m is from 1 to
 4. 12. A composition according to claim 7in which the ethylenic unsaturated group Y is H₂C═CR—CO-A-, in which Ris hydrogen or methyl and A is NH or O.
 13. A composition according toclaim 7 in which the zwitterionic monomer is2-methacryloyloxyethyl-2′-trimethylammonium ethyl phosphate inner salt.14. A composition according to claim 7 in which the ethylenicallyunsaturated monomers include comonomer.
 15. A composition according toclaim 14 in which the comonomer has the general formula IX

in which R¹⁹ is selected from the group consisting of hydrogen, halogen,C₁₋₄ alkyl and groups COOR²³ in which R²³ is hydrogen and C₁₋₄ alkyl;R²⁰ is selected from the group consisting of hydrogen, halogen and C₁₋₄alkyl; R²¹ is selected from the group consisting of hydrogen, halogen,C₁₋₄ alkyl and groups COOR²³ provided that R¹⁹ and R²¹ are not bothCOOR²³; and R²² is selected from the group consisting of C₁₋₁₀ alkyl, aC₁₋₂₀ alkoxycarbonyl, a mono- or di-(C₁₋₂₀ alkyl) amino carbonyl, aC₆₋₂₀ aryl, C₇₋₂₀ aralkyl, C₆₋₂₀ aryloxycarbonyl,C₁₋₂₀-aralkyloxycarbonyl, C₆₋₂₀ arylamino carbonyl, C₇₋₂₀ aralkyl-amino,hydroxyl and C₂₋₁₀ acyloxy groups, any of which may have one or moresubstituents selected from the group consisting of halogen atoms,alkoxy, oligo-alkoxy, aryloxy, acyloxy, acylamino, amine, carboxyl,sulphonyl, phosphoryl, phosphino, zwitterionic, hydroxyl,vinyloxycarbonyl, and reactive silyl and silyloxy groups; or R²² and R²¹or R²² and R²⁰ may together form —CONR²⁴CO in which R²⁴ is a C₁₋₂₀ alkylgroup.
 16. A composition according to claim 1 in which the ionic blockis formed of ethylenically unsaturated monomers including an ionicmonomer of general formula VIY¹B¹Q  VI in which Y¹ is selected from the group consisting ofH₂C═CR¹⁴—CO-A⁸-, H₂C═CR¹⁴—C₆H₄-A⁹-, H₂C═CR¹⁴—CH₂A¹⁰,R¹⁶O—CO—CR¹⁴═CR¹⁴—CO—O, R¹⁴CH═CH—CO—O—, R¹⁴CH═C(COOR¹⁶)CH₂—CO—O,

A⁸ is —O— or NR¹⁵; A⁹ is selected from the group consisting of a bond,(CH₂)_(q)A¹⁰ and (CH₂)_(q) SO₃— in which q is 1 to 12; A¹⁰ is selectedfrom the group consisting of a bond, —O—, O—CO—, CO—O, CO—NR¹⁵—,—NR¹⁵—CO, O—CO—NR¹⁵, and NR¹⁵—CO—O—; R¹⁴ is hydrogen or C₁₋₄ alkyl; R¹⁵is hydrogen, C₁₋₄₋ alkyl or B¹Q; R¹⁶ is hydrogen or C₁₋₄ alkyl; B¹ isselected from the group consisting of a bond, straight and branchedalkanediyl groups, alkylene oxaalkylene groups and alkylene(oligooxalkylene) groups, optionally containing one or more fluorinesubstituents; and Q is an ionic or ionisable moiety.
 17. A compositionaccording to claim 16 in which Q is selected from groups having theformula —NR¹⁷ _(p), —PR¹⁷ _(p) and SR¹⁷ _(r), in which p is 2 or 3, r is1 or 2, the groups R¹⁷ are the same or different and each is selectedfrom the group consisting of hydrogen, C₁₋₂₄ alkyl and aryl, or two ofthe groups R¹⁷ together with the heteroatom to which they are attachedform a 5 to 7 membered heterocyclic ring or three R¹⁷ groups togetherwith the heteroatom to which they are attached form a 5 to 7 memberedheteroaromatic ring, either of which rings may be fused to another 5 to7 membered saturated or unsaturated ring, and any of the R¹⁷ groups maybe substituted by amino or hydroxyl groups or halogen atoms.
 18. Acomposition according to claim 17 in which Q is —NR¹⁷ ₂ where each R¹⁷is the same and is C₁₋₁₂-alkyl.
 19. A composition according to claim 16in which B¹ is a C_(2-6-alkanediyl).
 20. A composition according toclaim 16 in which the ethylenically unsaturated monomers include acomonomer.
 21. A composition according to claim 20 in which thecomonomer has the general formula IX

in which R¹⁹ is selected from the group consisting of hydrogen, halogen,C₁₋₄ alkyl and groups COOR²³ in which R²³ is hydrogen or C₁₋₄ alkyl; R²⁰is selected from the group consisting of hydrogen, halogen and C₁₋₄alkyl; R²¹ is selected from the group consisting of hydrogen, halogen,C₁₋₄ alkyl and groups COOR²³ provided that R¹⁹ and R²¹ are not bothCOOR²³; and R²² is selected from the group consisting of C₁₋₁₀ alkyl, aC₁₋₂₀ alkoxycarbonyl, mono- and di-(C₁₋₂₀ alkyl) amino carbonyl, C₆₋₂₀aryl, C₇₋₂₀ aralkyl, C₆₋₂₀ aryloxycarbonyl, C₁₋₂₀-aralkyloxycarbonyl,C₆₋₂₀ arylamino carbonyl, C₇₋₂₀ aralkyl-amino, hydroxyl and C₂₋₁₀acyloxy groups, any of which may have one or more substituents selectedfrom the group consisting of halogen atoms, alkoxy, oligo-alkoxy,aryloxy, acyloxy, acylamino, amine, carboxyl, suiphonyl, phosphoryl,phosphino, zwitterionic, hydroxyl, vinyloxycarbonyl and reactive silyland silyloxy groups; or R²² and R²¹ or R²² and R²⁰ may together form—CONR²⁴CO in which R²⁴ is a C₁₋₂₀ alkyl group.
 22. A compositionaccording to claim 1 in which at least one of the blocks has apolydispersity of molecular weight less than 2.0.
 23. A compositionaccording to claim 1 in which the degree of polymerisation of the ionicblock is in the range 5 to 2000 and the degree of polymerisation of thezwitterionic block is in the range 2 to 1000 and in which the ratio ofthe degrees of polymerisation of the ionic block to the zwitterionicblock is in the range 1:5 to 10:1.
 24. A composition according to claim23 in which the degree of polymerisation of the ionic block is in therange 10 to 250, the degree of polymerisation of the zwitterionic blockis in the range 5 to 100 and the ratio of the degrees of polymerisationof the ionic block to the zwitterionic block is in the range 1:1 to 5:1.25. A composition according to claim 1 in which at least one of theblocks is formed by a living radical polymerisation process.
 26. Acomposition according to claim 25 in which the living radicalpolymerisation process is a group or atom transfer polymerisationprocess.
 27. A composition according to claim 1 in which the relativeamounts of biologically active compound and polymer are in the range 1:5to 10:1 based on equivalents of the polymer to active compound chargedgroups.
 28. A composition according to claim 27 in which the saidrelative amounts are in the range 1:2 to 5:2.
 29. A compositionaccording to claim 1 in which the biologically active compound ispolyanionic.
 30. A composition comprising a block copolymer having anoverall ionic charge and associated with the polymer a biologicallyactive compound having a charge opposite that of the polymer wherein theblock copolymer comprises at least one zwitterionic block which haspendant zwitterionic groups and at least one ionic block which compriseionic groups to confer said overall charge, wherein the biologicallyactive compound is anionic and wherein the ionic block is formed ofethylenically unsaturated monomers including an ionic monomer of generalformula VI:Y¹B¹Q  VI in which Y¹ is selected from the group consisting ofH₂C═CR¹⁴—CO-A⁸-, H₂C═CR¹⁴—C₆H₄-A⁹-, H₂C═CR¹⁴—CH₂A¹⁰,R¹⁶O—CO—CR¹⁴═CR¹⁴—CO—O, R¹⁴CH═CH—CO—O—, R¹⁴CH═C(COOR¹⁶)CH₂—CO—O,

A⁸ is —O— or NR¹⁵; A⁹ is selected from the group consisting of a bond,(CH₂)_(q)A¹⁰ and (CH₂)_(q) SO₃— in which q is 1 to 12; A¹⁰ is selectedfrom the group consisting of a bond, —O—, O—CO—, CO—O, CO—NR¹⁵—,—NR¹⁵—CO, O—CO—NR¹⁵—, and NR¹⁵—CO—O—; R¹⁴ is hydrogen or C₁₋₄ alkyl; R¹⁵is hydrogen, C₁₋₄ alkyl or B¹Q; R¹⁶ is hydrogen or C₁₋₄ alkyl; B¹ isselected from the group consisting of a bond, straight and branchedalkanediyl groups, alkylene oxaalkylene groups, and alkylene(oligooxalkylene) groups, optionally containing one or more fluorinesubstituents; and Q is —NR¹⁷ ₂ where each R¹⁷ is the same and is C₁₋₁₂alkyl.